Project Summary Osteocytes are multi-functional bone cells that maintain skeletal homeostasis. They reside in lacunae surrounded by matrix. Dendritic processes encased in canaliculi extend from osteocytes to form a network of channels needed for transport of nutrients and small molecules. Studies suggest osteocytes are capable of resorbing the surrounding matrix to maintain mineral ion homeostasis and regulate canalicular structure. During lactation, enlarged osteocyte lacunae are seen with enhanced osteocyte expression of genes classically expressed by osteoclasts to mediate bone resorption, including NFATc1 and its target genes cathepsin K and tartrate-resistant acid phosphatase. Metalloproteinases like MMP13 are also necessary for maintaining canalicular organization and regulating lacunar matrix resorption. Of note, mice lacking normal canalicular structure have increased osteocyte apoptosis. X-linked hypophosphatemia (XLH) is characterized by elevated serum fibroblast growth factor 23 (FGF23) levels, leading to decreased production of 1,25 dihydroxyvitamin D (1,25D) and hypophosphatemia. We have shown that tibiae and calvariae from the murine model of XLH (Hyp) have enlarged osteocyte lacunae and severely impaired canalicular organization accompanied by increased osteocyte apoptosis and enhanced osteocyte expression of bone resorption genes. Treatment of Hyp mice with 1,25D or a FGF23 blocking antibody, which increases serum 1,25D levels, similarly improves the lacuno-canalicular (LCN) structure, suggesting 1,25D acts directly on osteocytes to regulate LCN remodeling. To address this hypothesis in Aim I, the LCN phenotype will be characterized in mice lacking the vitamin D receptor in osteocytes (VDRf/f;DMP1Cre+) that have normal mineral ion homeostasis and skeletal mineralization. Because mice lacking the sodium phosphate transporter 2a (Npt2aKO) have hypophosphatemia with inappropriately normal serum 1,25D levels on D14 and high serum 1,25D levels by D30, the LCN phenotype will be characterized in Npt2aKO mice to determine if hypophosphatemia in the absence of elevated serum PTH stimulates LCN remodeling and if 1,25D maintains LCN structure in setting of the low serum phosphate. Studies proposed in Aim II will define a role for NFAT signaling in LCN remodeling and will determine whether 1,25D regulates signaling molecules of the NFAT pathway in osteocytes. In addition to defining a role for 1,25D in modulating LCN remodeling, these studies will contribute to the understanding of the impaired skeletal integrity and aberrant LCN remodeling seen in diseases like XLH as well as physiologic states like lactation, aging, and vitamin D deficiency.